The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases (RTKs) is important in many human cancers, and is the target of several FDA-approved therapeutic agents. Currently-used EGFR-targeted antibody therapeutics were specifically selected to inhibit EGF binding and EGF-dependent activation of the receptor. However, it is now clear that many EGFR-dependent cancers arise from oncogenic mutations in EGFR that promote ligand-independent signaling - which may not be blocked by antibodies such as cetuximab. Studies of oncogenically activated EGFR from lung cancer patients have been key in advancing understanding of how the EGFR kinase domain is regulated. An increasing number of mutations are being identified in the extracellular region of all EGFR family members, in glioblastoma and other cancers. These open up a similar opportunity for fully understanding regulation of the extracellular region - an important goal since current structure-based models of EGFR receptor activation do not explain (or predict) which of these somatic mutations will activate the receptors. In this proposal we first ask how oncogenic mutations in the EGFR extracellular region can promote ligand-independent activation of the receptor, and also refine our understanding of how the extracellular region of EGFR is maintained in its inactive state. In a second aim, we ask what are the most effective strategies to inhibit oncogenically-activated EGFR using antibody therapeutics. Agents (such as cetuximab) that simply block ligand binding may not be effective inhibitors of receptors that are activated by oncogenic mutation. We will compare the ability of different EGFR-targeted antibodies and novel single chain antibody-based agents (VHH domains) to inhibit constitutive (ligand-independent) signaling by mutated EGFR variants found in cancers. We will combine cellular studies with in vitro and structural analyses of the EGFR extracellular region to investigate these questions. Our Specific Aims are: 1: To understand which intramolecular interactions contribute to extracellular autoinhibition of EGFR, and how they are reversed by ligand binding and somatic mutations found in cancer patients. 2: To test the hypothesis that known oncogenic mutations in EGFR differentially affect the inhibitory activity of EGFR-targeted antibodies and of novel VHH domain agents. In sum, these studies will shed important new light on the mechanisms through which oncogenic mutations cause constitutive activation of EGFR and other ErbB receptors, while also identifying new antibodies that are uniquely able to block ligand-independent activation of EGFR mutants. Such agents will be of high priority for future clinical development.